PDBsum entry 1gxk

Chromosome segregation

PDB id

1gxk

Contents

			Protein chains

					158 a.a. *

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Molecular architecture of smc proteins and the yeast cohesin complex.

Authors

C.H.Haering, J.Löwe, A.Hochwagen, K.Nasmyth.

Ref.

Mol Cell, 2002, 9, 773-788. [DOI no: 10.1016/S1097-2765(02)00515-4]

PubMed id

11983169

Abstract

Sister chromatids are held together by the multisubunit cohesin complex, which contains two SMC (Smc1 and Smc3) and two non-SMC (Scc1 and Scc3) proteins. The crystal structure of a bacterial SMC "hinge" region along with EM studies and biochemical experiments on yeast Smc1 and Smc3 proteins show that SMC protamers fold up individually into rod-shaped molecules. A 45 nm long intramolecular coiled coil separates the hinge region from the ATPase-containing "head" domain. Smc1 and Smc3 bind to each other via heterotypic interactions between their hinges to form a V-shaped heterodimer. The two heads of the V-shaped dimer are connected by different ends of the cleavable Scc1 subunit. Cohesin therefore forms a large proteinaceous loop within which sister chromatids might be entrapped after DNA replication.



	Figure 3. Figure 3. Smc1/3 Dimerization Specificity Is Solely Conferred by the Hinge Domains(A) The hinge domain is necessary for Smc1/3 dimerization. Smc1Δhinge or Smc1 were coexpressed with His[6]Smc3 in insect cells and subjected to a pull-down assay on Ni^2+-NTA. The presence of Smc1Δhinge or Smc1 in input (I), unbound (U), and bound (B) fractions was probed by immunoblotting with an antibody specific to the N terminus of Smc1 (upper panel) and the efficiency of Smc3 binding to the resin with anti-His antibody (lower panel).(B) Only molecules with opposite hinge domains can dimerize. Smc1, HA[3]Smc3, or Smc1hinge3 were coexpressed in insect cells with either His[6]Smc3 or His[6]Smc3hinge1, and protein association of each combination was assayed as in (A).(C) Electron micrographs of the Smc3hinge1/Smc3 dimer. The His[6]Smc3hinge1/ HA[3]Smc3 dimer was purified from insect cells over Ni^2+-NTA and gel filtration. Proteins in the peak fraction from the gel filtration were rotary shadowed with a 2 nm platinum layer and visualized in the electron microscope.(D) The hinge domain of Smc3 is sufficient for binding to Smc1. N-terminal, hinge, and C-terminal globular domains of Smc3 were coexpressed with Smc1 in insect cells as HA[3]-tagged proteins. The globular domains were immunoprecipitated and their ability to pull down Smc1 was tested by immunoblotting for Smc1 (upper panel). Full-length HA[3]Smc3 was used as a positive control. In addition, the association of the HA[3]Smc3hinge domain with Smc1hinge3 was tested. In all experiments, the efficiency of the HA[3]-immunoprecipitation was tested by blotting against the HA[3] epitope (lower panel).(E) The Smc3hinge domain binds Smc1 as tightly as the full-length Smc3 protein does. HA[3]Smc3 or the HA[3]Smc3hinge domain produced in insect cells was bound to a CM5 sensor chip on the BIAcore system via a monoclonal anti-HA antibody attached to covalently linked anti-mouse Fc γ-specific antibody. Insect cell extracts containing defined concentrations of Smc1 as the ligand (five dilutions, ranging from 20 nM to 200 nM) were floated over the bound analytes, and association and dissociation kinetics were recorded. For each dilution, the data were fitted using a 1:1 Langmuir binding model with drifting baseline and corrected for unspecific binding to uninfected insect cell extracts. The average association and dissociation rate constants (k[a] and k[d], respectively) are displayed and used to calculate the equilibrium binding constant (K[A]). Low average values of χ^2 indicate the accuracy of the fit and the suitability of the 1:1 binding model, the variation coefficients ν for the binding constants show the consistency of the measurements over the ligand dilution range.(F) Crystal structure of the hinge domain from Thermotoga maritima SMC protein (construct HTMC9, residues 473-685). Ribbon drawing of the hinge domain dimer, showing two stretches of antiparallel coiled coil (yellow and green). The orientation is essentially the same as in Figure 1B. The coiled coil segments are formed by residues from the same chain, resulting in an intramolecular coiled coil arrangement for SMC proteins. The structure shown was re-solved in spacegroup C2 by seleno-methionine substitution and MAD at 3.0 Å resolution.(G) Architecture of SMC proteins. The intramolecular coiled coil results in the two arms being formed by separate chains with the hinge domains holding the two arms together. The coiled coil segments have been modeled using standard geometry and the crystal structures of the hinge and head domains have been described here and elsewhere (Löwe et al., 2001). Figure prepared using MOLSCRIPT (Kraulis P.J., 1991).		Figure 8. Figure 8. Model of the Yeast Cohesin Complex(A) Smc1 and Smc3 form a heterodimer with intramolecular coiled coils. Scc1 bridges the head domains of Smc1 and Smc3 and links them to Scc3. For comparison, a schematic 10 nm chromatin fiber of DNA wrapped around nucleosomes and a DNA double helix are shown in scale to the Smc1/3 ring.(B) Hypothetical “embrace” model of how the cohesin complex might confer sister chromatid cohesion. Before the commencement of replication, the cohesin complex is loaded onto DNA. The arms of the Smc1/3 molecules embrace the DNA, thereby forming a ring of approx. 40 nm diameter. The head domains of Smc1 and Smc3 are locked together by Scc1. Now, cohesion might be generated as the replication fork passes through the ring, entrapping both sister chromatids inside. At the metaphase to anaphase transition, Scc1 is cleaved by separase, thereby opening the lock of the Smc1/3 head domains. The ring opens and sister chromatids can be pulled to opposite spindle poles.

PROCHECK

	Headers